Pneumocystis jirovecii pneumonia remains a major cause of infection in patients with AIDS. Lung inflammation during Pneumocystis pneumonia (PcP) strongly contributes to lung injury and death. Anti- inflammatory corticosteroids (in addition to antibiotics) significantly improve outcome during PcP, but are associated with further immune suppression and co-infection. Numerous studies demonstrate that Pneumocystis (Pc) cell wall components including ?-glucans interact with alveolar macrophages and epithelial cells to stimulate the release of cytokines and chemokines that promote inflammatory cell recruitment into the lungs. ?-glucans are homopolymers of D-glucose consisting of ?-1,3 linked chains with variable amounts and configurations of ?-1,6 linked glucose side chains. Most studies have focused on unfractionated cell wall ?- glucans or on ?-1,3 glucans during inflammatory responses to fungi. However, recent studies strongly indicate that actually the ?-1,6 glucan exposed side chains of fungal cell walls are responsible for a considerable amount, if not the major portion, of host inflammatory responses. To date, the role of ?-1,6 glucans have not been studied in any pathogenic fungi such as Pneumocystis, only in S. cerevisiae models. To address ?-1,6 glucans in Pc, over the last funding period we demonstrated that P. carinii cell walls indeed contain significant ?-1,6 glucans, in addition to the ?-1,3 glucan generated by PcGsc1 in Pc. We further have shown that ?-1,6 glucans strongly stimulate inflammatory signaling in lung cells, and have identified the ?-1,6 glucan generating machinery in Pneumocystis mediated by the PcKre6 enzyme. We also observed that host cell membrane lactosylceramide strongly participates in inflammatory activation in response to Pc ? -1,6 glucans. Despite this progress, considerable gaps still exist in our understanding of these important host-organism interactions. In particular, the regulation of Pc ? -1,6 (as opposed to ? -1,3) glucan in the organism is not well understood. It is also unknown how Pc cell wall generation responds to the lung micro-environment. In addition, host receptors and signaling pathways that specifically mediate inflammatory signaling to Pc ? -1,6 compared to ? -1,3 glucans are not yet defined. Finally, strategies to ameliorate the associated exuberant and damaging lung inflammation during Pneumocystis infection are not well developed. On this basis, we hypothesize that Pneumocystis ? -1,6 glucans strongly activate deleterious inflammatory signaling in epithelial cells and macrophages through lactosylceramide mediated MAPK signaling with subsequent cytokine/chemokine generation. These concepts will be addressed through three independent, but closely interrelated, aims. In Aim 1, we will evaluate the P. carinii ? -1,6 glucan synthetic machinery (compared to the ?-1,3) by characterizing the PcKre6 and PcGsc1 synthetases, their regulation over the life cycle of Pc, and their responses to environmental stimuli including interactions with host epithelial cells. We will further quantify the relative content of ?-1,6 as opposed to ?-1,3 glucans both in P. carinii and P. jirovecii samples, and will study the regional localization of ?-1,6 and ?-1,3 glucans in the Pc cyst wall. Under Aim 2, we will evaluate mechanisms by which whole Pneumocystis and isolated Pc ?-1,6 vs. ?-1,3 glucans separately activate MAPK signaling in macrophages and epithelial cells, resulting in translocation of NF-?B, and release of inflammatory cytokines (TNF?) and chemokines (MIP- 2). We will also elaborate the role of membrane glycosphingolipids in mediating cellular activation, as well as defining the roles of the known general glucan receptors in triggering these specific interactions. We will further measure host humoral responses to Pc ?-1,6 and ?-1,3 glucans both in the immunosuppressed rat models, as well as in archival human BAL and blood samples. Finally, in Aim 3, we will evaluate the utility of glycospingolipid inhibitors and newly identified ?-1,6 gluca synthase inhibitors as prophylactic and therapeutic regimens for PcP, defining their impact on lung inflammation, gas exchange, and Pc burden. Better understanding the roles of ?-1,6 and ?-1,3 glucan related innate inflammatory signaling during PcP will better define mechanisms of lung injury during infection. Moreover, this proposal also presents the potential to develop new agents with strongly beneficial effects on organism-driven lung inflammation during this important cause of morbidity and mortality in patients with AIDS.